INDUCIBLE EXPRESSION OF GREEN FLUORESCENT PROTEIN WITHIN CHANNEL CATFISH CELLS BY A CECROPIN GENE PROMOTER

The activity of an insect promoter of the cecropin B gene (Cec B) was investigated using green fluorescent protein (gfp) as a reporter in ce lls of channel catfish (Ictalurus punctatus). The expression vector pQ Z-1 containing the Cec B promoter and a modified gfp cDNA sequence was delivered by lipofection to three catfish cell types: fibroblast and leukocyte cell lines, and primary cultures of leukocytes. No resistanc e genes were included in the vector for selection of GFP-expressing ce lls. The GFP mRNA was detected in all three cell types with 5 to 10 ti mes higher concentrations observed in leukocytes than in fibroblasts. Expression was enhanced with the addition of irradiated Flavobacterium columnare (7.0 x 10(6) cells/ml) or Escherichia coli LPS (125 mu g/ml ). Quantitative RT-PCR showed GFP mRNA reached maximum levels 24 h aft er bacterial challenge in fibroblast cells, and at 10-12h after LPS ch allenge in fibroblasts and leukocytes. The number of fibroblasts expre ssing GFP increased by 0.8%, and the average of green fluorescence int ensity increased by 52.8%, whereas the increase in leukocytes was 0.13 % in cell number and 3.4% in fluorescence intensity. These results sug gest that the transcription of the Cec B promoter in channel catfish c ells exhibited an inducible pattern and could be placed under the cont rol of the immune system (in vivo). The mechanisms for endogenous acti vation of the Cec B promoter and for production of gfp RNA in unchalle nged cells remain to be studied. (C) 1998 Elsevier Science B.V. All ri ghts reserved.